Mammalian haploid embryonic stem cells

ABSTRACT

The invention relates to mammalian haploid embryonic stem cells and methods for the production of such stem cells. The inventions also relates to a cell culture and a cell line of mammalian haploid embryonic stem cells.

FIELD OF THE INVENTION

The present invention relates to a mammalian haploid embryonic stem celland methods for the production of the mammalian haploid embryonic stemcell. In an additional aspect, the invention also relates to a cellculture comprising the mammalian embryonic haploid stem cell and a stemcell line obtainable by proliferation of the stem cell. In a furtheraspect, the invention relates to the use of the haploid embryonic stemcells in genetic screening.

BACKGROUND OF THE INVENTION

Genetic screening is an important tool to identify new genes or mutantalleles of known genes that underlie biological processes. Increasinglystem cells, particularly embryonic stem cells, have been used in thesescreens. Stem cells are characterised by two important properties, whichare of value in genetic screening. Firstly, they have an ability toproliferate in an undifferentiated state for prolonged periods of time.Secondly, stem cells are pluripotent. This means that they are capableof differentiating into any cell of the mesoderm, ectoderm or endoderm.As a result, pluripotent cells can develop into any cell of the body.

It is at present however, very difficult to screen for recessivemutations in mammalian cells. The main reason is that mammalian cellsare diploid, meaning that each cell has two copies of each gene. As aresult, the phenotypic traits of heterozygous recessive mutations aremasked by the second copy of the gene.

A solution to screen for recessive mutations is to use a haploidmammalian embryonic stem cell. Haploid stem cells are stem cells that incontrast to diploid stem cells possess only one copy of each gene. As aresult, the phenotypic traits of recessive mutations are essentiallyunmasked, and as a result the underlying genes can be easily identifiedand studied.

Haploid embryonic stem cells have been obtained from fish as describedin Yi and Hong (2009). In this paper, the authors developed haploidembryonic stem cell lines from the medaka fish (Oryzias latipes).

However, notably, at present, no such cells have been obtained frommammals and fish haploid embryonic stem cells can not be used as asubstitute. Unlike placental mammals, fish are oviparous animals andhave no placentation. As a result, embryonic development differssubstantially from fish to mammals. Furthermore, key features such assex determination and dosage compensation are also regulated differentlyand implantation, placentation and genomic imprinting are all absent inMedaka fish.

The use of a near-haploid tumour cell line for genetic screening hasbeen described by Carette et al., (2009). In this paper, insertionalmutagenesis was used to generate null alleles in a human cell linehaploid for all chromosomes except chromosome 8. Therefore, the cellline described in this paper was not a true haploid. Furthermore, as thecell line carried genetic rearrangements and a transformed phenotype,its usefulness in genetic screening in developmental system is limited.

A number of reports have also found near-haploid cells in a variety ofhuman tumours, Nonetheless, except for one case, no near-haploid celllines have been derived from these tumours (Sukov et al., 2010).

Finally, Kaufman et al. (1983) describe the production of pluripotentcell lines from haploid embryos from parthenogenetically activatedoocytes. However, the cell lines even at early passages of the cellswere diploid not haploid.

Accordingly, there exists a need to produce mammalian haploid embryonicstem cells. Such cells would enable genetic screening for recessivemutations in a developmentally relevant context. For example, it isenvisaged that such cells would be an important tool in identifying newgenes involved in signalling pathways, developmental decisions and cellcycle regulation. Moreover, there is a need to produce haploid stemcells without tumour-derived mutations, genomic rearrangements oroncogenes. Stem cells with such characteristics are obtained when cellsare derived from tumour cells. Accordingly, there exists a need todevelop mammalian embryonic stem cells with a normal karyotype.

SUMMARY OF THE INVENTION

According to a first aspect of the present invention, there is provideda mammalian haploid embryonic stem cell, wherein the stem cell ispluripotent, capable of proliferation, and can maintain a haploidkaryotype during proliferation in culture. After haploid ES cells havebeen established they can be maintained in a wide range of conditions.Haploid ES cells can be cultured in standard ES cell medium conditionsusually including LIF and fetal calf serum or Knock-out serumreplacement (Invitrogen; 10828028) or chemically defined media using 2iinhibitors.

In certain embodiments, the haploid ES cells appear to have a smallersize than diploid ES cells.

In a preferred aspect, the stem cell can maintain a haploid karyotypefor at least 15, preferably 20, and more preferably at least 25 passagesof the stem cell. A passage is the degree of subculturing of a cell.

A further aspect of the invention provides a method for the productionof a mammalian embryonic stem cell, wherein the method comprises thefollowing steps—

-   -   activating isolated oocytes in vitro to produce haploid embryos;    -   culturing the activated embryos to the 8-cell, morula or        blastocyst stage;    -   removing the zona pellucida;    -   isolating the inner cell mass of the activated embryos; and    -   further culturing the inner cell mass to derive a haploid        embryonic stem cell.

The method may further comprise the step of flow sorting based on DNAcontent, to isolate haploid ES cells.

The in vitro activation may comprise incubation in M16 medium, strontiumchloride and EGTA. In a further preferred embodiment, the incubationtime is no more than 5 hours, preferably 1 to 3 hours. M16 medium may beobtained from Sigma; Cat. no. M7292. Potential other means for oocyteactivation include activation with an electric pulse and by usingcalcium ionophores such as ionomycine.

In a further aspect, the cultured activated embryo is preferablycultured with kinase inhibitors. The kinase inhibitors may be selectedfrom threonine/tyrosine kinase inhibitors and/or glycogen synthasekinase 3β inhibitors. In a preferred embodiment, the kinase inhibitorsare PD0325901 and CHIR99021 (CHIR99021 from Stemgent Catalog #04-0004;PD0325901 from Stemgent Catalog #04-0006). The culture will typicallyinclude fibroblast feeder cells.

Other culturing methods may include:

a) High glucose DMEM medium (PAA, Cat. No. E15-009) or Knockout™ D-MEM(Invitrogen, Catalog#10829018) with 15% KnockOut™ Serum Replacement(Invitrogen, Catalog#10828028) supplemented with Glutamin (Invitrogen),beta mercapto ethanol (Sigma), penicillin-streptomycine (Invitrogen),non-essential amino acids (Invitrogen) and 500 units per milliliterrecombinant mouse LIF (home made).b) It is possible that conditions as a) but with 15% fetal calf serum(PAA) instead of 15% KnockOut™ Serum Replacement would also beeffective.

Potential other culture methods which may be suitable include:

c) Serumfree medium with BMP and LIF (commercially available fromMillipore, ESGRO Complete PLUS Clonal Grade Medium, Millipore Cat No.SF001-500P)

d) iSTEM media (Stem Cells, Inc, Cat. No. SCS-SF-ES-01), this is acommercial formulation of the 2i media but does not contain LIF.

e) A combination of 2i culture conditions and KnockOut™ SerumReplacement (Invitrogen) such as the one used in Hanna et al, 2010,PNAS, 107(20):9222-9227

The zona pellucida is preferably removed using acidic Tyrode's solution(Millipore; MR-004-D). Oocytes are incubated in Acidic Tyrode's solutionuntil zona pellucida is dissolved. This usually takes 30 seconds up toone minute.

In a further aspect, the trophectoderm layer of the blastocyst ispreferably removed using specific antibodies (immunosurgery) prior toisolation of the inner cell mass. For example, after removal of the zonaembryos are incubated with Anti-Mouse Serum antibody produced in rabbit(Sigma, M5774). After washing the trophectoderm layer is lysed usingcomplement (rat serum, home made or commercial such as Complement serafrom guinea pig, Sigma S1639). Immunosurgery is not required when EScells are derived using 2i conditions. The procedure is described inManipulating the Mouse Embryo, A LABORATORY MANUAL, Second Ed., Hogan,B. et al., 1994, Cold Spring Harbour Laboratory Press, ISBN0-87969-384-3).

The cells of the inner cell mass may be cultured using either fibroblastfeeders in serum-free media, cytokines and kinase inhibitors or usinghigh glucose media supplemented with serum or a serum replacement andcytokines. In a more preferred aspect, the serum-free media is N2B27medium. The cytokine is preferably LIF (Leukemia Inhibitory Factor). Inanother embodiment the high glucose media is DMEM and the serumreplacement is KnockOut™ Serum Replacement (KSR).

Cell growth may be monitored by microscopy and cell clumps aredissociated using trypsin (0.25%, Invitrogen; 15090046) or Accutase(Invitrogen; A110501). The inner cell mass outgrowth and embryonic stemcells grow as a colony morphology. Haploid embryonic stem cells areobtained when many colonies are obtained in the culture dish which growrapidly. The cells are passaged every 3 to 4 days, or every 2 to 3 dayswhen growth speeds up. We believe that haploid ES cells are alreadypresent at the first passage, but it takes about 4 to 5 passages toexpand haploid ES cells from a single ICM to a confluent 6 well sizeddish with about 3×10⁶ cells.

Haploid stem cells may be further be enriched from the culture usingcell sorting. Preferably, living cells are stained. Any type of cellularDNA stain may be used. Preferably, the stain may be a fluorescent stainsuch as a Hoechst stain (Hoechst 33342 at a concentration of 1 microgramper milliliter). Haploid cells can be sorted using a cell sorter,preferably a fluorescence-activated cell sorter. Potential other DNAstains include VYBRANT® DYECYCLE™ STAINS for living cells (Invitrogen,Molecular probes), DRAQ5® (4084, Cell Signaling Technology) andNuclear-ID™ Red DNA Stain (ENZ-52406, ENZO LIFE SCIENCES INTERNATIONAL,INC.) Other enrichment methods may include separation by cell size suchas elutriation or subcloning from single cells.

An additional aspect of the invention provides a cell culture comprisingthe mammalian haploid embryonic stem cells. The culture conditions formaintaining haploid ES cells include all conditions for derivation(above). Culture in DMEM with 15% serum and LIF has also been successfulfor maintaining haploid ES cells.

A further aspect of the invention provides a mammalian haploid embryonicstem cell line obtained by proliferation of the mammalian embryonic stemcell. The embryonic stem cell line is pluripotent, capable ofproliferation and has a stable haploid karyotype during proliferation.

Another aspect of the invention provides the use of the mammalianembryonic stem cell line in genetic screening. In a preferredembodiment, the genetic screen is a genetic forward screen. We havesuccessfully performed a forward genetic screen in the haploid ES cellsas follows. A pool of haploid ES cells is exposed to a mutagen such asan insertional vector. Cells containing mutations can be selected forexample using a selection marker of the insertional mutagen. The pool ofmutant haploid cells can optionally now be diploidized as mutations willbe in a homozygous configuration. Selection of desired mutations can beachieved by appropriate metabolic conditions, reporter genes combinedwith cell sorting or by phenotypic characteristics including celladhesion and cell surface markers. We have performed a pilot screen as aproof of principle experiment for genes involved in the mismatch repairpathway. In this screen a Piggybac transposon derived vector was used asthe insertional mutagen. Selection for cells with mutations in genesrequired for mismatch repair was achieved using 2-amino-6-mercaptopurine(Sigma, 6-TG), a substance that is toxic to cells proficient in mismatchrepair. The gene mutations were identified by a Splinkerette PCRstrategy. This screen identified the two known autosomal mismatch repairgenes Msh2 and Fanci (see below) and demonstrates the principleapplication of haploid mouse ES cells for genetic screening.

Potential other mutagens include: a) Genetrap vectors such as attenuatedviruses; b) Chemical mutagens; c) Physical methods for mutagenesis suchas ionizing irradiation.

Potential other methods for identifying mutations include: a) Reportergene activity combined with cell sorting; b) Reporter gene activitycombined with antibiotic resistance; c) Cell surface molecules combinedwith appropriate methods for cell isolation such as antibody based; d)Metabolic conditions; e) Exposure to biological agents such as pathogensfor instance for identifying host cell factors or receptors for viruses.

Potential other methods for gene identification include: a) 5 prime racefor identification of trapped genes; b) Genome wide expressionprofiling; c) DNA sequencing; d) directed screening for mutations inspecific genes or gene sets such as by DNA sequencing or PCR.

A benefit of using mouse ES cells is the availability of a large numberof genetic modifications that have been introduced into the mouse genomeand specific mutations can be readily generated. Therefore, geneticmodification of haploid ES cell lines or derivation of haploid ES cellslines from genetically modified mouse strains can be utilized to enablecomplex screens or make screens more specific. Potential uses are (1)the incorporation of sensitizing mutations for finding synthetic lethalcombinations of genes for uncovering parallel or redundant pathways and(2) the introduction of appropriate selection markers such as reportergenes for pathway activity that enable or support the isolation ofdesired mutations.

Potential other uses for haploid ES cells include:

a) Haploid ES cells can be introduced into mice. One way of doing thisis by diploidizing them in culture to obtain parthenogenetic diploid EScells. These can be introduced via blastocyst injection, morulaaggregation or 8-cell injection into chimeric mice. Germlinetransmission of parthenogenetic mouse ES cells has been reported andpresents a route for introducing mutations identified in a screen intomice for further study.b) Haploid ES cells can be used for cell fusion. Two different haploidcells can be combined to give a diploid cell. Similarly, triploid cellsresult from fusion of a haploid ES cell with a diploid cell. Potentialpartners for fusion with haploid ES cells are other haploid ES cells forinstance to combine two genetic modifications. This could be useful fortesting genetic interactions. Other potential applications could be thefusion of naturally haploid cells such as gametes with haploid ES cellsto derive diploid cells.c) Haploid ES cells can be used to derive differentiated cells. HaploidES cells can differentiate but in the course of differentiation theymostly become diploid.

In a preferred aspect, the mammal is a rodent, preferably a mouse (Musmusculus). In a more preferred aspect, the strain of the mouse isCBA/B6. However, a number of different strains of mouse can be used.This would allow different genetic modifications to be introduced, suchas selectable marker genes.

Potential other mouse strains include:

a) inbred mouse strains such as 129Sv or C57/BL6

b) outbred mouse strains—We have successfully derived haploid ES cellsfrom a mixed background containing a genetic modification of the Xistgene locus (Xist2LOX).

c) Genetically modified mouse strains such as strains carrying genedeletions or transgenes. Especially, mouse strains containing reportergenes (including antibiotic resistance or GFP) such as Oct4 or Rex1promoter driven GFP reporter strains could be used for screening. Inaddition reporters for cell signalling pathways could be introduced fromthe mouse strain such as a TOPflash reporter for Wnt signalling pathwayactivity. In addition mutations for sensitizing the genetic backgroundcan be introduced by deriving haploid ES cells from mice carrying genedeletions. An important aspect of haploid mouse ES cells is the abilityto realize the potential of a large number of preexisting mousemutations that could be utilized to screen for specific components inmammalian signaling, cell cycle, and metabolic pathways.

ES cells with similar properties as mouse ES cells have been describedfrom rats and rabbits (Telugu et al, 2010, The International Journal ofDevelopmental Biology 54: 1703-1711). The method of the presentinvention could be applied to these mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described, by way of example only,with reference to the accompanying drawings, in which

FIG. 1 shows a strategy for the derivation of the mammalian haploidembryonic stem cells.

FIGS. 2A and 2B show the characterisation of a haploid mouse embryonicstem (ES) cell line. FIG. 2A shows three representative pictures ofmetaphase spreads of a haploid mouse ES cell line showing a haploid setof 20 chromosomes. DNA was stained with DAPI. FIG. 2B shows a FACSanalysis of fixed ES cells after DNA staining with propidium iodide.Position of haploid (1n), diploid (2n), and tetraploid (4n) DNA contentis indicated. The haploid line (upper panel) shows a predominantly 1nDNA content with some G2 cells after DNA replication showing as 2n. Thecontamination with diploid cells is low as judged from the 4n peakcorresponding to G2 cells. A diploid control ES cell line (lower panel)shows a 2n G1 peak and a 4n G2 peak.

FIG. 3 shows immunofluorescence staining of haploid and control diploidES cells for detecting Nanog, a marker of pluripotent ES cells, andGata4, an early ES differentiation marker. Colonies of both haploid andcontrol diploid ES cells express the pluripotency marker Nanog but donot express markers that are indicative of differentiation.

FIG. 4 shows analytic flow profiles after DNA staining with PI for 129Svderived diploid control embryonic stem cells, the haploid H129-1 ES cellline at passage 6 and at passage 12 after two rounds of sorting the 1npeak.

FIGS. 5A-5H show derivation of haploid ESCs. Flow analysis of DNA afterPI staining of (FIG. 5A) diploid control ESCs, (FIG. 5B) haploid ESCline HAP-1 at passage 7 (p7) and (FIG. 5C) HAP-1 (p11) after sorting atp7. (FIG. 5D) Colony morphology of haploid ESCs (HAP-1). (FIG. 5E AND5F) Chromosome spreads of HAP-3 (FIG. 5E) and HAP-1 (FIG. 5F), (Scalebar=10 μm). (FIG. 5G) CGH analysis of HAP-1 and HAP-2 ESCs and controlmale CBA kidney DNA. Relative copy number is plotted at 200 kbresolution using a log 2 scale. Genomic positions indicated by blue bars(top) are enlarged at 40 kb resolution in (FIG. 5H); CBA control(black), HAP-1 (red) and HAP-2 (green).

FIGS. 6A-6D show expression analysis of haploid ESCs. (FIG. 6A)Immunofluorescence shows Nanog protein (red) in haploid (HAP-1) anddiploid ESCs, and Gata4 (green) in differentiated cells (Scale bar=10μm). (FIG. 6B) Expression of pluripotency markers in haploid and diploid(set to 1) ESCs by real time PCR. Error bars represent standarddeviation (n=3). (FIG. 6C) Scatter plot showing log 2 transformedaverage expression values from gene expression profiles of 3 haploid(HAP-1, HAP-2 and HTG-1) and three diploid J1 ESC lines for 45,001 probesets (r is the Pearson correlation coefficient; red lines indicate2-fold up- and down-regulation). (FIG. 6D) Diagram of more than 2-foldup- and down-regulated genes in haploid ESCs.

FIGS. 7A-7D show developmental potential of haploid ESCs. (FIG. 7A) GFPmarked haploid HAP-2 ESCs (p18) contribute to chimeric embryos at E12.5.6 out of 9 embryos showed GFP contribution. A GFP negative embryo isshown as a control (below). (FIG. 7B) Representative flow analyses ofDNA content of all cells (above) and GFP positive cells (below)extracted from a chimeric E12.5 embryo are shown. All 6 embryos gavesimilar results. (FIG. 7C) Live born chimeric mice were obtained fromGFP marked HAP-2 ESCs. (FIG. 7D) Chimeric mice obtained from injectionof HAP-1 ESCs into C57BU6 blastocysts (black) show coat colourcontribution from the ESCs (agouti).

DETAILED DESCRIPTION OF THE INVENTION Example 1

Oocyte Activation

Oocytes were isolated in the morning from mouse oviducts after hormonalinduction of ovulation. Ovulation was induced by intraperitonealinjection of PMS and hCG 48 hours later (this superovulation procedureis described in Manipulating the mouse embryo, ISBN 0-87969-384-3). Apotential other method for obtaining unfertilized oocytes could bethrough natural matings with vasectomised males.

Activation of oocytes was carried out by incubation in M16 medium towhich 5 mM (millimolar) SrCl₂ and 2 mM EGTA were added. Incubation timesof 1 to 3 hour were found to be efficient. Shorter incubation led tolower activation rates whereas 5 hour incubation appeared to affectsubsequent development. After activation oocytes were cultured in groupsof 50 using microdrop culture. For this 80 microliter drops of M16medium were overlayed with mineral oil. All culture media werepreequilibrated and incubation was at 37° C., 5% CO₂ in a tissue cultureincubator (SANYO). Blastocyst stage embryos were obtained at day 3 to 4and used for ES cell derivation.

Activation of freshly ovulated oocytes in vitro is mediated by inductionof Calcium (Ca) waves. This can be achieved by prolonged culture inmedium without Ca. The addition of Strontium (Sr) has been used toincrease activation rates and enhance the development of diploidparthenogenetic embryos (Bianchi et al., 2010). Complexing the Ca withEGTA has made it possible to use preformulated media in combination withSr for efficient activation of mouse oocytes (Kishigami and Wakayama,2007). Similar protocols are established based on permeabilization ofthe oocyte membrane for Ca by ionomycine treatment. These protocols aremore relevant for other mammalian species including human where Sr doesnot lead to efficient oocyte activation.

ES Cell Derivation in Chemically Defined Medium

For derivation of ES cells in chemically defined medium 8-cell embryoswere cultured for two days in M16 supplemented with the followinginhibitors: 1 μM PD0325901 and 3 μM CHIR99021 (M16-2i). 2i is acomposition that enhances the efficiency of ES cell line derivation andthereby reduces the number of embryos required (see, for example,Nature. 2008 May 22; 453(7194):519-23. The ground state of embryonicstem cell self-renewal. Ying QL, Wray J, Nichols J, Batlle-Morera L,Doble B, Woodgett J, Cohen P, Smith A).

This step expanded the inner cell mass of the blastocyst. The zonapellucida was then removed with acidic Tyrode's solution (Hogan et al.,1994). Optionally, the trophectoderm was removed by immunosurgery.Isolated inner cell masses or embryos were then cultured on mitoticallyinactivated embryonic fibroblast feeders in chemically defined N2B27medium containing above 2i inhibitors and LIF (N2B27-2i plus LIF). LIFis a cytokine that inhibits embryonic stem cell differentiation andstimulates stem cell growth. Outgrowths were passaged repeatedly every 3to 4 days until ES cell lines emerged. In 2i ES cell lines emerged afterthe first passage.

ES cell culture in defined medium with signal inhibition (Ying et al.,2008) can be used to derive ES cells from recalcitrant mouse strains(Nichols et al., 2009) and rats (Buehr et al., 2008). Recently, similarapproaches have led to a culture of an embryonic stem cell state fromhumans that have similar properties with mouse ES cells (Hanna et al.,2010).

ES Cell Derivation in Standard Conditions

For ES cell derivation the zona pellucida was removed from morula andblastocyst stage embryos with acidic Tyrode's solution (Hogan et al.,1994). After removal of the trophectoderm by immunosurgery (Hogan etal., 1994) the inner cell mass was explanted and cultured in highglucose DMEM supplemented with fetal bovine serum or serum replacement(KSR) and LIF. Outgrowths were passaged every 3-4 days until ES celllines were obtained.

Enrichment for Cells with 1n Content Using Cell Sorting

HOECHST 33342 stained living cells were sorted using a DAKO MoFlo highspeed sorter. Cells with a haploid (1n) DNA content were selected.Diploid cell lines (2n) were used as controls. For analysis the FlowJoFlow Cytometry Analysis Software was used (Tree Star Inc.).

Additionally, ES cell lines can be subcloned.

Characterization of Haploid ES Cell Lines

DNA content was investigated by analytical FACS (Fluorescence ActivatedCell Sorting). Karyotypes were identified by metaphase chromosomespreads. Haploid ES cell lines display a characteristic colonymorphology and growth rate comparable to diploid mouse ES cells. Inaddition these cells express markers of mouse ES cells (FIG. 3).

Forward Genetic Screen to Identify Genes Involved in Mismatch Repair.

This proof of principle screen was designed according to a screenperformed previously (Genome Res. 2009 April; 19(4):667-73. Epub 2009Feb. 20. A piggyBac transposon-based genome-wide library ofinsertionally mutated Blm-deficient murine ES cells. Wang W, Bradley A,Huang Y).

5×10⁶ haploid ES cells were co-transfected with a gene trap cassetteembedded in a PiggyBac vector and a vector carrying the PiggyBactransposase gene. Integration of the gene trap cassette into atranscribed locus brings a puromycin resistance cassette under controlof the endogenous promoter. This allows selection for successfulintegration of gene trap cassettes into the genome using puromycin(Sigma; P8833) at a concentration of 2 microgram per milliliter. Thepool of puromycin resistant haploid ES cells was used for furtherscreening. Cells were seeded at a density of 1.5×10⁶ per 15 cm plate andselection for mutations in MMR genes was performed using 2 μM,2-amino-6-mercaptopurine (Sigma, 6-TG). Selection was initiated 24 hafter plating and continued for 8d. Colonies were picked and expandedbefore analysis. PiggyBac integrations were mapped by Splinkerette PCR.(Mikkers, H., et al (2002) High-throughput retroviral tagging toidentify components of specific signaling pathways in cancer. Nat.Genet. 32:153-159.) Results are shown in table 2.

Conclusion

The above results describe the successful production of a mouse haploidembryonic stem cell. As discussed previously, to date there has been noprior description of a mammalian haploid cell and cell line. Indeed, itwas previously believed that the derivation of such cells wasimpossible. Moreover, the above-described methods allow haploidembryonic stem cells to be derived from unfertilised oocytes. As aresult, such cells do not contain tumour derived mutations, genomicrearrangements or oncogenes. We therefore describe a means for derivingembryonic stem cells with a normal haploid karyotype. Haploid ES cellsprovide a platform for highly efficient forward genetic screening inmammalian cells. Further, we believe that haploid ES cells aremaintained most purely if they are sorted every few passages. Haploid EScells also have a tendency to diploidize, which could enable thegeneration of developmental models.

TABLE 1 Derivation of haploid mouse ES cell lines ES cell max. ES celllines with haploid derivation genetic Oocytes Nr of lines haploidcontribution protocol background activated blastocysts obtainedcontribution (%) 2i/immuno- CBA/B6 132 30 27 6 40 surgery 2i CBA/B6 2210 5 1 15 2i mixed 50 32 3 3 60 KSR/immuno- CBA/B6 273 48 22 6 10surgery

TABLE 2 Forward genetic screen in haploid ES cells to identify genesinvolved in mismatch repair cell line number genes identified known MMRgenes 1 Msh2 2 Fanci 3 Msh2 novel MMR candidates or false positives 4Zfp462, Lypd1, nlgn1, Slc44a3 5 Acbd6, Lypd1, nlgn1 6 Usp14, Atl1, 7SK 7Wdr40a, Sorbs2

Example 2

Derivation of Haploid Embryonic Stem Cells from the 129Sv Inbred MouseStrain

140 oocytes were collected from superovulated 129Sv female mice andactivated using strontium chloride and EGTA in M16 medium for 2 hours.Embryos were cultured in M16 medium until the blastocyst stage. 13blastocysts were obtained, the zona was removed and the inner cellmasses were cultured in 2i medium in the presence of LIF and BSA. Form atotal of 10 embryonic stem cell lines obtained, 3 showed a substantialcontent of cells with a haploid genome equivalent. The haploid cellcontent was estimated between 40% and 60%. Sorting of the haploid 1npeak allowed the establishment of pure haploid 129Sv mouse embryonicstem cells cultures. FIG. 4 shows analytic flow profiles after DNAstaining with PI for 129Sv derived diploid control embryonic stem cells,the haploid H129-1 ES cell line at passage 6 and at passage 12 after tworounds of sorting the 1n peak.

Example 3

Most animals are diploid but haploid-only and male-haplo species havebeen described¹. Diploid genomes of complex organisms limit geneticapproaches in biomedical model species such as in mice. To overcome thisproblem experimental induction of haploidy has been used in fish^(2,3).In contrast, haploidy appears less compatible with development inmammals^(4,5). Here we describe haploid mouse embryonic stem cells andshow their application in forward genetic screening.

Experimentally induced haploid development in zebrafish has beenutilized for genetic screening². Recently, haploid pluripotent celllines from medaka fish have also been established³. In contrast to fish,haploidy is not compatible with development in mammals. Although haploidcells have been observed in egg cylinder stage parthenogenetic mouseembryos⁶ the majority of cells in surviving embryos become diploid.Previous attempts to establish pluripotent stem cell lines from haploidembryos have resulted in the isolation of parthenogenetic embryonic stemcells (ESCs) with a diploid karyotype⁴. These studies reported thedevelopment of apparently normal haploid mouse blastocysts with adefined inner cell mass (ICM)^(4,5). In order to investigate the haploidICM, we cultured haploid mouse blastocysts in chemically defined mediumwith inhibitors of mitogen activated protein kinase kinase (MEK) andglycogen synthase kinase 3 (GSK3). This 2i medium⁷ has previously beenused for isolating ESCs from recalcitrant mouse strains⁸ and rats⁹ andmay help to maintain certain characteristics of early mouse epiblastcells^(10,11).

We generated haploid mouse embryos by activation of unfertilized oocytesisolated from superovulated B6CBAF1 hybrid female mice using strontiumchloride. After culture in M16 medium 30 blastocysts (22%) were obtainedfrom 132 activated oocytes and used for ESC derivation. After removal ofthe zona and trophectoderm, ICMs were cultured in gelatinized 96 welldishes in 2i medium in the presence of LIF. 27 ESC lines were obtained(93%). Individual ESC lines were expanded and their DNA content wasanalysed by flow analysis using diploid ESCs as controls (FIGS. 5A and5B). In 6 ESC lines, at least 10% of the cells had a haploid DNA contentand the proportion of haploid cells could reach a conservativelyestimated 60% (FIG. 5B). Further enrichment was achieved by flow sortingof cells with a haploid DNA content after staining with HOECHST 33342(FIG. 5C). This allowed expansion of haploid ESC lines for over 35passages.

We further tested the requirements for deriving haploid mouse ESCs(Table 3). These experiments showed that removal of the trophectoderm byimmunosurgery was not essential. Haploid ESCs could also be establishedusing DMEM medium supplemented with Knockout Serum Replacement (KSR) andLIF showing that derivation without kinase inhibitors is possible (Table3 and FIGS. 8A and 8B). We further succeeded in isolating haploid ESCsfrom the 129Sv inbred mouse strain and two genetically modified mouselines. In the latter several alleles had been bred to homozygosity andmaintained on a mixed genetic background for several generations (Table3, and FIGS. 9A and 9B). In summary, we derived 25 haploid ESC lines in7 independent experiments. Haploid ESC cultures could also be maintainedon feeders in serum containing DMEM supplemented with LIF.

Haploid ESCs exhibited a typical mouse ESC colony morphology (FIG. 5D).Chromosome spreads showed 20 chromosomes corresponding to the haploidmouse chromosome set (FIGS. 5E and 5F). For further characterizing thegenetic integrity we performed comparative genomic hybridization (CGH)of 4 haploid ESC lines and control DNA from the CBA strain and the mixedtransgenic mouse line from which HTG-1 and HTG-2 ESCs were derived(FIGS. 5G and 5H and FIGS. 10A, 10B, and 11). Copy number variations(CNVs) that were detected in the genome of haploid ESC lines were alsopresent in the strains of origin (FIG. 16). Albeit some CNVs appearedhaploid ESC specific, inspection of the actual signals (FIG. 11)suggested that these CNVs were also present in the CBA or HTG controlDNAs but not detected with the threshold applied. CNVs between theC57BL6 and CBA strain of mice were consistent with a previously reportedanalysis¹². Taken together these data show that haploid ESCs maintainedan intact haploid genome without amplifications or losses.

On the molecular level, haploid ESCs expressed pluripotency markersincluding Oct4, Rex1, Klf4, Sox2 and Nanog (FIGS. 6A and 6B).Genome-wide expression analysis showed a high correlation (Pearsoncorrelation coefficient r=0.97 over all genes) between haploid ESCs andcontrol diploid male ESCs (FIG. 6C and FIG. 12). In haploid ESCs 279 and194 genes were more than 2-fold up- or down-regulated (p<0.05),respectively (FIG. 17). Among these, 99 X-linked genes wereoverexpressed and 4 Y-linked genes were lost in haploid ESCs consistentwith different sex chromosome constitutions (FIG. 6D). Thus, haploidESCs largely maintain a mouse ESC transcription profile.

This prompted us to investigate the developmental potential of haploidESCs. For this we introduced a piggyBac transposon vector for expressinggreen fluorescent protein (GFP) into HAP-2 ESCs. Flow sorting of cellsfor GFP fluorescence and DNA staining with Hoechst 33342 yielded ahaploid ESC population that expressed GFP at high level showing that ahaploid genome content was maintained during the transfection procedure(FIGS. 13A and 13B). GFP marked haploid ESCs contributed substantiallyto chimeric embryos when injected into C57BL/6 blastocysts (FIG. 7A).The great majority of GFP positive cells extracted from chimeric embryoshad a diploid DNA content (FIG. 7B) indicating that haploid ESCscontributed extensively to development after diploidization. We alsoobtained 2 male and 2 female live born chimeras with a substantialcontribution from haploid ESCs (FIG. 7C). These mice developed normallywith apparent coat colour chimerism. Similar results were obtained withthe HAP-1 and HTG-2 ESCs (FIG. 7D and FIG. 14A). Furthermore, thediploid fraction of HAP-2 ESCs at passage 31 could be differentiatedinto Nestin positive cells following a neural in vitro differentiationprotocol¹³ (FIG. 14B). Taken together these findings demonstrate thathaploid ESCs maintain a wide differentiation potential. These chimericmice have transmitted the GFP transgene from the GFP transgenic HAP-1haploid embryonic stem cell line to two of their offspring. This isevidence for the ability of introducing mutations or geneticmodifications into mice via haploid ES cells.

To investigate the utility of haploid ESCs for genetic screening weperformed a pilot screen for mismatch repair genes following apreviously published strategy¹⁴. For this, 5×10⁶ haploid ESCs wereco-transfected with a gene trap piggyBac transposon vector (FIG. 15A)and a plasmid for expressing an optimized piggyBac transposase¹⁵. Genetrap insertions were selected with puromycin. A pool of 1×10⁷ cells wasthen cultured in the presence of 2-amino-6-mercaptopurine (6-TG) whichis toxic to mismatch repair proficient cells. After 8 days 20 6-TGresistant colonies were isolated and the integration sites were mappedusing Splinkerette PCR¹⁶. Of 7 clones analysed we identified twoindependent insertions in Msh2 and one in Hprt (FIG. 15B). Msh2 is aknown mismatch repair gene and Hprt is required for converting 6-TG intoa toxic metabolite¹⁴. Thus, identification of mutations in autosomalgenes was possible suggesting a potential for haploid ESCs in forwardgenetic screening in mammals.

The difficulty in obtaining haploid ESC lines in previous attempts mightbe explained by aberrant gene regulation such as aberrant dosagecompensation and genomic imprinting. However, diploid ESCs from mouseand human parthenogenetic embryos have been established^(17,18).Misregulation of X inactivation has been observed to some extent inhaploid mouse embryos⁵ and has also been shown to reduce the efficiencyof producing cloned mice¹⁹. Thus, it is conceivable that X inactivationis initiated aberrantly in haploid embryos during some ESC derivationprocedures. Direct capture of naive pluripotent cells from ICMoutgrowths as accentuated by the use of 2i conditions¹¹ could havecontributed to the success of our study.

Previously, near-haploid cells have been observed in human tumours (forliterature review see²⁰) and a near-haploid human tumour derived cellline has been described^(21,22). These tumour cells carry genomicrearrangements and mutations that might stabilize the haploid genome. Aninteresting aspect of haploid ESCs is their developmental potential. Wehave observed rapid diploidization when haploid ESCs differentiate. Theresulting diploid parthenogenetic cells can contribute to development²³.It is interesting to speculate whether differentiated haploid lineagescan be generated perhaps through suppression of X inactivation andwhether it is possible to derive haploid human ESCs.

Methods Summary

For the derivation of haploid ESCs mouse oocytes were activated in M16medium as described²⁴. ESC culture in chemically defined 2i medium hasbeen described previously^(7,8). Cell sorting for DNA content wasperformed after staining with 15 μg/ml Hoechst 33342 (Invitrogen) on aMoFlo flow sorter (Beckman Coulter) selecting the haploid 1n peak. Foranalytic flow profiles cells were fixed in ethanol, RNase treated, andstained with propidium iodide (PI). For karyotype analysis cells werearrested in metaphase with demecolcine (Sigma). After incubation inhypotonic KCl buffer cells were fixed in methanol-acetic acid (3:1) andchromosome spreads were prepared and stained with DAPI. RNA wasextracted using the RNeasy Kit (Quiagen). Transcription profiles weregenerated using Affymetrix GeneChip 430.2 arrays. Sample preparation,hybridization, and basic data analysis were performed by Imagenes(Berlin, Germany). Further analysis was performed using the GenespringGX software (Agilent). For CGH analysis genomic DNA was isolated fromhaploid ESC lines and hybridized to NimbleGen 3×720K whole-genome tilingarrays by Imagenes (Berlin, Germany) using C57BU6 kidney DNA as areference. For chimera experiments GFP labelled HAP-1 (p29), HAP-2 (p18)and HTG-2 (p23) ESCs were injected into C57BU6 host blastocysts. Liveborn chimaeras were analysed for expression of GFP at postnatal day 2.Genetic screening was performed following a previously publishedstrategy²⁵. In brief, HAP-1 ESCs were co-transfected with 2 μg piggyBactransposase expression vector¹⁵ and 1 μg piggyBac gene trap vector(FIGS. 15A and 15B) using Lipofectamine 2000 (Invitrogen). Selection fortransposon insertions was performed using 2 μg/ml puromycin for 8 days.1×10⁷ puromycin resistant ESCs were plated in two 15 cm dishes andmutations in mismatch repair genes were selected using 0.3 μg/ml 6-TG(Sigma). piggyBac integration sites in seven 6-TG resistant clones weremapped by Splinkerette PCR¹⁶.

References for Example 3

-   1. Otto, S. P. & Jarne, P. Evolution. Haploids—hapless or happening?    Science 292, 2441-3 (2001).-   2. Wiellette, E. et al. Combined haploid and insertional mutation    screen in the zebrafish. Genesis 40, 231-40 (2004).-   3. Yi, M., Hong, N. & Hong, Y. Generation of medaka fish haploid    embryonic stem cells. Science 326, 430-3 (2009).-   4. Kaufman, M. H., Robertson, E. J., Handyside, A. H. & Evans, M. J.    Establishment of pluripotential cell lines from haploid mouse    embryos. J Embryol Exp Morphol 73, 249-61 (1983).-   5. Latham, K. E., Akutsu, H., Patel, B. & Yanagimachi, R. Comparison    of gene expression during preimplantation development between    diploid and haploid mouse embryos. Biol Reprod 67, 386-92 (2002).-   6. Kaufman, M. H. Chromosome analysis of early postimplantation    presumptive haploid parthenogenetic mouse embryos. J Embryol Exp    Morphol 45, 85-91 (1978).-   7. Ying, Q. L. et al. The ground state of embryonic stem cell    self-renewal. Nature 453, 519-23 (2008).-   8. Nichols, J. et al. Validated germline-competent embryonic stem    cell lines from nonobese diabetic mice. Nat Med 15, 814-8 (2009).-   9. Buehr, M. et al. Capture of authentic embryonic stem cells from    rat blastocysts. Cell 135, 1287-98 (2008).-   10. Nichols, J., Silva, J., Roode, M. & Smith, A. Suppression of Erk    signalling promotes ground state pluripotency in the mouse embryo.    Development 136, 3215-22 (2009).-   11. Nichols, J. & Smith, A. The origin and identity of embryonic    stem cells. Development 138, 3-8 (2011).-   12. Cutler, G., Marshall, L. A., Chin, N., Baribault, H. &    Kassner, P. D. Significant gene content variation characterizes the    genomes of inbred mouse strains. Genome Res 17, 1743-54 (2007).-   13. Pollard, S. M., Benchoua, A. & Lowell, S, Neural stem cells,    neurons, and glia. Methods Enzymol 418, 151-69 (2006).-   14. Li, M. A., Pettitt, S. J., Yusa, K. & Bradley, A. Genome-wide    forward genetic screens in mouse ES cells. Methods Enzymol 477,    217-42 (2010).-   15. Cadinanos, J. & Bradley, A. Generation of an inducible and    optimized piggyBac transposon system. Nucleic Acids Res 35, e87    (2007).-   16. Mikkers, H. et al. High-throughput retroviral tagging to    identify components of specific signaling pathways in cancer. Nat    Genet. 32, 153-9 (2002).-   17. Mai, Q. et al. Derivation of human embryonic stem cell lines    from parthenogenetic blastocysts. Cell Res 17, 1008-19 (2007).-   18. Revazova, E. S. et al. Patient-specific stem cell lines derived    from human parthenogenetic blastocysts. Cloning Stem Cells 9, 432-49    (2007).-   19. Inoue, K. et al. Impeding Xist expression from the active X    chromosome improves mouse somatic cell nuclear transfer. Science    330, 496-9 (2010).-   20. Sukov, W. R. et al. Nearly identical near-haploid karyotype in a    peritoneal mesothelioma and a retroperitoneal malignant peripheral    nerve sheath tumor. Cancer Genet Cytogenet 202, 123-8 (2010).-   21. Kotecki, M., Reddy, P. S. & Cochran, B. H. Isolation and    characterization of a near-haploid human cell line. Exp Cell Res    252, 273-80 (1999).-   22. Carette, J. E. et al. Haploid genetic screens in human cells    identify host factors used by pathogens. Science 326, 1231-5 (2009).-   23. Jiang, H. et al. Activation of paternally expressed imprinted    genes in newly derived germline-competent mouse parthenogenetic    embryonic stem cell lines. Cell Res 17, 792-803 (2007).-   24. Kishigami, S. & Wakayama, T. Efficient strontium-induced    activation of mouse oocytes in standard culture media by chelating    calcium. J Reprod Dev 53, 1207-15 (2007).-   25. Guo, G., Wang, W. & Bradley, A. Mismatch repair genes identified    using genetic screens in Blm-deficient embryonic stem cells. Nature    429, 891-5 (2004).

TABLE 3 Derivation of haploid mouse ESC lines Names of ESC ESC lineswith haploid Exp. lines used genetic Oocytes number of ESC linescontribution (max. % No derivation protocol in study backgroundactivated blastocysts obtained haploid before sorting) 12i/immunosurgery HAP-1 to 6 ^(†) B6CBAF1 132 30 27 6 (>60%) 2 2i HAP-7B6CBAF1 22 10 5 1 (>15%) 3 2i HTG-1 to 3 ^(‡) mixed TG* 50 32 3 3 (>90%)4 KSR/immunosurgery HAP-8 to 13 B6CBAF1 273 48 22 6 (>10%) 5 2i H129B6-1to 5 129B6F1 250 37 8 5 (>10%) 6 2i HTX-1 mixed TG** 70 11 1 1 (>40%) 72i H129-1 to 3 129Sv 140 13 10 3 (>60%) ^(†) Contribution to chimericmice was confirmed for the HAP-1 and HAP-2 haploid ESC lines. ^(‡)Contribution to chimeric mice was confirmed for the HTG-1 haploid ESCline. *Derived from ROSA26^(nlsrtTA) LC1 Xist^(2LOX) homozygous femalemice. **Derived from ROSA26^(nlsrtTA) tetOPXist homozygous femalemice³⁰.Methods for Example 3Derivation of Haploid ESCs

Oocytes were isolated from superovulated females and activated in M16medium using mM strontium chloride and 2 mM EGTA as described²⁴. Embryoswere subsequently cultured in M16 or KSOM medium microdrops covered bymineral oil. Under these conditions around 80% of oocytes reached the2-cell stage on the next morning. Thereafter development ofpreimplantation embryos was variable with a large number of embryosshowing unequal sized blastomeres or unusual embryo morphology. Removalof the zona, immunosurgery for removal of the trophectoderm and ESCderivation was performed as described previously^(7,8). ESCs werecultured in chemically defined 2i medium plus LIF as described^(7,8)with minor modifications. 2i medium was supplemented with non essentialamino acids and 0.35% BSA fraction V. Culture of ESCs on feeders wasperformed as previously described²⁶. Knockout serum replacement (KSR)was obtained from Invitrogen. Cell sorting for DNA content was performedafter staining with 15 μg/ml Hoechst 33342 (Invitrogen) on a MoFlo flowsorter (Beckman Coulter). The haploid 1n peak was purified. Diploidcells did arise in cultures to various extents in all ESC lines.Periodic purification by flow sorting every four to five passagesallowed us to maintain cultures containing a great majority of haploidESCs in all cases. Analytic flow profiles of DNA content were recordedafter fixation of the cells in ethanol, RNase digestion, and stainingwith propidium iodide (PI) on a Cyan analyser (Beckman Coulter). Forkaryotype analysis cells were arrested in metaphase using demecolcine(Sigma). After incubation in hypotonic KCl buffer cells were fixed inmethanol-acetic acid (3:1) and chromosome spreads were prepared andstained with DAPI. Immunostaining was performed as described²⁷ usingNanog (Abcam; 1:100), Oct4 (Santa Cruz; 1:100), Nestin (DevelopmentalStudies Hybridoma bank, Iowa City; 1:30) and Gata4 (Santa Cruz; 1:200)antibodies.

Microarray Analysis

RNA from biological triplicates of diploid ESCs and three independentlyderived haploid ESCs (HAP-1p21, HAP-2 p24, HTG-1 p11) was extractedusing the RNeasy kit (Quiagen). Gene expression analysis on AffymetrixGeneChip 430 2.0 arrays was performed by Imagenes Ges.m.b.H. (Berlin,Germany). Additional gene expression profiles of neural progenitors,mesodermal progenitors and mouse embryonic fibroblasts (MEF) wereobtained from a previously published dataset (GEO accession numberGSE12982²⁸). The data was analysed using Genespring GX software (AgilentTechnologies). Data were normalized using the RMA algorithm. Listsshowing differentially regulated genes (>2 fold change; p<0.05) areprovided in FIG. 17. p-values were established by an unpaired t testfollowed by FDR adjustment by the Benjamini Hochberg method.Hierarchical clustering was performed based on the Euclidean distancesand complete linkage analysis. The relatedness of transcription profileswas determined by calculating the Pearson correlation coefficient (r).DNA samples for comparative genomic hybridization (CGH) experiments wereextracted and sent to Imagenes Ges.m.b.H. (Berlin, Germany) for CGHanalysis using NimbleGen 3×720K mouse whole-genome tiling arrays with anaverage probe spacing of 3.5 kb. Adult male C57BL/6 kidney DNA was usedas a reference. A genomic overview of these analyses is presented inFIG. 5G and FIGS. 10A and 10B at 200 kb resolution and selected zoomedin regions at 40 kb resolution. The complete data set at 40 kbresolution is included in FIG. 11.

Accession of Datasets

Gene expression and CGH data sets can be accessed as the GEO referenceseries GSE30879(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30879). Thisseries includes the GSE30744 (Expression analysis of haploid and diploidES cells in 2i medium) and the GSE30749 (CGH analysis of haploid EScells) data sets.

Quantitative Gene Expression Analysis

RNA was extracted using the RNeasy kit (Quiagen) and converted into cDNAusing the Quantitect reverse transcription kit (Quiagen). Real time PCRwas performed on a StepOnePlus machine (Applied Biosystems) using theFast Sybr green master mix (Applied Biosystems) and previously publishedprimers²⁷. The ddCt method was used for quantification of geneexpression. Expression levels were normalized to L32 ribosomal proteinmRNA and values in diploid control ESCs were set to 1.

Embryo Analysis

Haploid ESCs were co-transfected with a piggyBac vector carrying aCAG-GFP—IRES-hygro transgene and a piggyback transposase expressionplasmid. Stable integrants were selected using 150 μg/ml Hygromycin for7 days. The haploid fraction of HAP-1 (p29), HAP-2 (p18) and HTG-2 (p23)GFP positive cells were purified by flow sorting (FIGS. 13A and 13B).GFP labelled ESCs were expanded and injected into C57BL/6 hostblastocysts which were transferred to recipient females. Embryos wereanalysed at E9.5 and E12.5. Dissociation to single cells was performedby incubation in 0.25% Trypsin/EDTA for 15 min. Prior to PI stainingcells were fixed in 4% PFA and permeabilized in PBS/0.25% Triton X-100.Live born chimeras were analysed at postnatal day 2 (P2) for expressionof GFP using UV illumination. Images were obtained using a CanonPowershot S5 IS camera with a FHS/EF-3GY2 filter (BLS). All mouseexperiments were conducted in accordance with institutional guidelinesof the University of Cambridge. All necessary UK home office licenseswere in place.

Gene Trap Screen

The screen was performed based on a previously published protocol²⁵.5×10⁶ HAP-1 ESCs were co-transfected with 2 μg piggyBac transposaseplasmid¹⁵ and 1 μg piggyBac gene trap vector (FIG. 15A) usingLipofectamine 2000. piggyBac insertions into expressed genes wereselected with 2 μg/ml puromycin for 8 days. 1×10⁷ ESCs corresponding toapproximately 5,000 puromycin resistant colonies were then plated ontotwo 15 cm dishes. Selection for mismatch deficient integrants wasperformed using 0.3 μg/ml 6-TG (Sigma). 20 colonies were picked andpiggyBac integration sites of seven clones were identified bySplinkerette PCR and mapped using iMapper²⁹.

References Used Only in the Methods Section

-   26. Wutz, A. & Jaenisch, R. A shift from reversible to irreversible    X inactivation is triggered during ES cell differentiation. Mol Cell    5, 695-705 (2000).-   27. Leeb, M. et al. Polycomb complexes act redundantly to repress    genomic repeats and genes. Genes Dev 24, 265-76 (2010).-   28. Shen, X. et al. EZH1 mediates methylation on histone H3 lysine    27 and complements EZH2 in maintaining stem cell identity and    executing pluripotency. Mol Cell 32, 491-502 (2008).-   29. Kong, J., Zhu, F., Stalker, J. & Adams, D. J. iMapper: a web    application for the automated analysis and mapping of insertional    mutagenesis sequence data against Ensembl genomes. Bioinformatics    24, 2923-5 (2008).-   30. Savarese, F., Flahndorfer, K., Jaenisch, R., Busslinger, M. &    Wutz, A. Hematopoietic precursor cells transiently reestablish    permissiveness for X inactivation. Mol Cell Bio126, 7167-77 (2006).

The invention claimed is:
 1. A mammalian haploid embryonic stem cellline, wherein the stem cell line is pluripotent, proliferates, andmaintains a haploid karyotype during proliferation in culture for aminimum of 45 days, wherein the cell line is cultured in conditionsselected from: a) a N2B27 chemically defined medium comprising LIF andone or more 2i inhibitor; b) a N2B27 chemically defined mediumcomprising 15% KnockOut Serum Replacement supplemented with Glutamin,betamercaptoethanol, penicillin-streptomycin, non-essential amino acids,and 500 units per milliliter recombinant mouse LIF; c) a N2B27chemically defined medium comprising 15% fetal calf serum supplementedwith Glutamin, betamercaptoethanol, penicillin-streptomycin,non-essential amino acids, and 500 units per milliliter recombinantmouse LIF; d) high glucose DMEM medium or KnockOut DMEM mediumcomprising 15% fetal calf serum supplemented with Glutamin,betamercaptoethanol, penicillin-streptomycin, non-essential amino acidsand 500 units per milliliter recombinant mouse LIF; or e) high glucoseDMEM medium or KnockOut DMEM medium comprising 15% KnockOut SerumReplacement supplemented with Glutamin, betamercaptoethanol,penicillin-streptomycin, non-essential amino acids, and 500 units permilliliter recombinant mouse LIF.
 2. The mammalian haploid embryonicstem cell line of claim 1, wherein the proliferation conditions furthercomprise a fibroblast feeder layer.
 3. The mammalian haploid embryonicstem cell line of claim 1, wherein the stem cell is capable ofmaintaining a haploid karyotype for at least 25 passages.
 4. Themammalian haploid embryonic stem cell line of claim 1, wherein themammalian haploid embryonic stem cell line is derived from Mus musculusof a CBA/B6 strain.
 5. A cell culture comprising the mammalian haploidembryonic stem cell line of claim
 1. 6. The mammalian haploid embryonicstem cell line of claim 1, wherein the mammalian haploid embryonic stemcell line is prepared according to the steps of activating one or moreisolated oocytes in vitro to produce a haploid embryo; culturing theactivated haploid embryo to an 8-cell stage, a morula stage, or ablastocyst stage; removing the zona pellucida from the activated haploidembryo; isolating the inner cell mass of the activated haploid embryo;and further culturing the inner cell mass to derive the mammalianhaploid embryonic stem cell line.